Con la finalidad de insertar los genes cry3Ca1 y cry7Aa1 en Ipomoea batatas (L.) Lam. cv. “Huachano” para conferir resistencia a Cylas puncticollis y C. brunneus “gorgojos del camote”, se desarrolló la transformación genética mediada por Agrobacterium tumefaciens, siguiendo dos protocolos de regeneración y transformación. Partiendo de 46 meristemos y 282 hojas con peciolo, se obtuvieron 8 eventos transgénicos de inserción completa del ADN - T (4 por cada protocolo de transformación) y un evento de inserción incompleta. Con una eficiencia de transformación de 8.70%, el protocolo a partir de meristemos demostró ser más eficaz en la obtención de eventos transgénicos de inserciones completas que el protocolo a partir de hojas con peciolo, con el cual se obtuvo una eficiencia de transformación de 1.42%. A su vez, los regenerantes fueron evaluados mediante pruebas in vitro (resistencia a kanamicina) y moleculares (PCR), con los análisis de PCR se confirmó que los 8 regenerantes callo positivos (resistentes a la prueba de kanamicina) también presentaron la inserción de los transgenes de interés “cry3Ca1 y cry7Aa1” y del gen marcador selector “nptII”. Asimismo, se determinó que 4 de los eventos transgénicos integraron secuencias externas al ADN - T “backbone” en el genoma de la planta, uno de los cuales, presentó la inserción del gen bacteriano “virD2”.With the purpose of inserting the genes cry3Ca1 and cry7Aa1 in Ipomoea batatas (L.) Lam. cv. "Huachano" to develop resistance to Cylas puncticollis and C. brunneus “sweet potato weevils”, the genetic transformation was developed using Agrobacterium tumefaciens, following two protocols of regeneration and transformation. Starting from 46 meristems and 282 leaves with petiole, 8 transgenic events were obtained with complete insertion of T - DNA (4 for each transformation protocol) and one event with incomplete insertion. With an efficiency of transformation of 8.70 %, the protocol of meristems proved to be more effective in obtaining transgenic events of complete inserts than the protocol of leaves with petiole, which obtained a efficiency of transformation of 1.42 %. The regenerants were evaluated by testing in vitro (resistance to kanamycin) and molecular (PCR), the PCR analysis confirmed that the 8 regenerants positive callus (resistant to kanamycin test) also presented the insertion of transgenes of interest “cry3Ca1 and cry7Aa1” and selection marker gene “nptII”. Likewise, 4 of the transgenic events integrated sequences outside the T - DNA “backbone” into the plant genome, one of whom, showed the insertion of the bacterial gene “virD2”.

Considerado um dos aspectos mais complexos da genética quantitativa, a epistasia tem sido ignorada pelos melhoristas nos estudos de herança dos caracteres, principalmente os da herança da resistência de plantas a insetos, que são de difícil obtenção. No milho, a principal praga de grãos é o Sitophilus zeamais (Coleoptera: Curculionidae), devido a sua capacidade de atacar grãos tanto no campo quanto em silos. Contudo, as estimativas dos componentes aditivo e de dominância envolvidos na herança dessa resistência podem estar viesadas pela presença do efeito da epistasia. Utilizando o delineamento triple testcross, os objetivos deste trabalho foram: (i) verificar a presença da epistasia para os caracteres relacionados à resistência do milho ao S. zeamais; (ii) estimar os efeitos epistáticos em cada planta F2; e (iii) estimar o efeito da interação epistasia x ambientes para estes caracteres. As 300 progênies de retrocruzamentos utilizadas nesse estudo foram avaliadas em dois ambientes no município de Piracicaba/SP, em delineamento alfa-látice 15 x 20, no esquema fatorial com duas repetições por ambiente. Os caracteres avaliados foram: número de insetos mortos (NM); número de insetos emergidos (EM); tempo médio de desenvolvimento dos insetos (TM); índice de suscetibilidade (IS) e perda percentual de massa seca dos grãos (PE). No ambiente E. E. Anhumas a presença da epistasia foi detectada para todos os caracteres; porém, no ambiente Caterpillar o efeito da epistasia não foi detectado para nenhum dos caracteres. Na análise conjunta, os efeitos epistáticos foram detectados para os caracteres NM, EM, IS e PE. O efeito da epistasia do tipo aditivo x dominante e/ou dominante x dominante foi mais importante para todos os caracteres que a epistasia do tipo aditiva x aditiva. A interação da epistasia com os ambientes foi significativa apenas para os caracteres TM e PE. Identificaram-se efeitos epistáticos bidirecionais significativos em plantas F2 para todos os caracteres no ambiente E. E. Anhumas e para os caracteres NM, EM, IS e PE na análise conjunta. O grande número de plantas F2 que apresentaram epistasia para mais de um caráter simultaneamente sugere a presença de epistasia pleiotrópica. As estimativas da variância aditiva e da interação aditiva com ambientes foram significativamente maiores que as das variâncias de dominância para a maioria dos caracteres. As magnitudes das estimativas dos coeficientes de herdabilidade para todos os caracteres variaram de baixas a medianas. A alta correlação genética entre os caracteres EM e PE sugere que o caráter PE, que é de difícil avaliação, pode ser selecionado indiretamente através do caráter EM que é de fácil avaliação para programas de melhoramento visando resistência ao S. zeamais. Os resultados obtidos na análise conjunta sugerem que, na população estudada, a epistasia constitui um componente importante da variância genética, de forma que as estimativas da variância aditiva, de dominância, graus médios de dominância e coeficientes de herdabilidade estão viesadas. / Considered one of the most complex components in quantitative genetics, the epistasis has been ignored by plant breeders, especially in inheritance studies of plant resistance, because the traits are laborious to evaluate. Sitophilus zeamais (Coleoptera: Curculionidae) is an important grain pest, due to its ability to attack in both field and silo conditions. However, the estimation of additive and dominance components in the inheritance of resistance to this pest may be biased due to epistatic effects. With the use of the triple test cross design, this research was aimed to: (i) verify the role of epistasis in the inheritance of maize resistance to S. zeamais, (ii) estimate the epistatic effects in the resistance traits of each F2 plant; (iii) estimate the epistasis x environment interaction. The 300 backcross progenies of this study were evaluated in two environments at Piracicaba, Brazil, in 2008/2009 growing season, using an alpha-lattice 15 x 20 design in a factorial arrangement with two replications per environment. The recorded traits were: number of dead weevils (NDW); number of emerged weevils (EW); mean development period (DP); index of susceptibility (IS) and the percentage of dry grain weight loss (DGWL). The epistatic effects were detected in Anhumas Experimental Station (AES) environment for all traits although they were absent in Caterpillar Experimental Station environment. In the combined analysis epistasis was detected for NDW, EW, IS and DGWL traits. The additive x dominance and/or dominance x dominance epistasis were more important than the additive x additive epistasis for all traits. The epistasis x environment interaction was significant only for traits DP and DGWL. Significant epistatic effects, which were not unidirectional, were detected in F2 plants for all traits in AES and for the traits NDW, EW, IS and DGWL in the combined analysis. Several F2 plants presented epistasis for more than one trait simultaneously suggesting the presence of pleiotropic epistasis. Estimates of additive, dominance and the additive by environment interaction variances were significant for all traits. Estimates of additive and additive by environment interaction variances were significantly higher than those of dominance variance for most of the traits. The magnitudes of the heritability coefficients estimates ranged from low to intermediate. The genetic correlation between EW and DGWL traits suggests that DGWL, which is difficult to evaluate, can be indirectly selected by the EW trait, which is easier to evaluate, in a breeding program. The results from the combined analysis suggests that, in the population studied, the epistasis is an important component of the genetic variance; therefore, estimates of additive, dominance variance, the average levels of dominance and heritability coefficients are biased.

Palms are ecologically important and charismatic trees of the tropics. They are important to the livelihood of local communities and are key resources for the frugivore community in tropical forests. These frugivores are in turn hunted by humans for food. This ecological connection between human, palms, and frugivores provides a unique setting to study how cultural and ecological components within this multitrophic interaction influences palm populations. In chapter 1, I explored the traditional and ecological knowledge behind the cultivation of palm-weevil larvae for food. I found the Joti people, cultivated two species of weevil-larvae differently, which also determined whether palms were logged before or after reproductive maturity. The cultivation of each weevil-larvae species therefore had a differential impact on palm populations. In chapter 2, I investigated how frugivores mediate interactions between two dominant and co-occuring palms in the Peruvian Amazonia-- Attalea phalerata and Astrocaryum murumuru. I found frugivores codispersed seeds of the two palm species, which contributed to aggregated spatial patterns of their juveniles. Spatial patterns suggested associations between heterospecific palms experienced lower density-dependent mortality than associations between conspecifics and this likely contributes to the coexistence of the two palm species in their early life-history. These findings highlight the importance of dispersers to species coexistence and suggest over-hunting can lead to shifts away from species codominance. In chapter 3, I examined the contribution of dispersal, distance-and density-dependent to spatial ecology of Attalea phalerata. Using microsatellite-based parentage analysis, I found high levels of seed movement mediated by frugivore dispersers. Despite this, I found dispersal limitation remains strong enough to cause spatial aggregation between offspring and parents. As individuals grew towards maturity, distance and density dependent mortality contributed to increasingly disaggregated patterns between older offspring cohorts of parents, non-parent adults, and siblings. These results provide a foundation for assessing the impacts of hunting on the spatial ecology of palm populations. In chapter 4, we characterized 14 microsatellite loci for A. phalerata that were used in the parentage analysis of chapter 3. These loci amplified reliably and were sufficiently polymorphic and will be useful for future studies addressing population-level questions for this species. / text

Considerado um dos aspectos mais complexos da genética quantitativa, a epistasia tem sido ignorada pelos melhoristas nos estudos de herança dos caracteres, principalmente os da herança da resistência de plantas a insetos, que são de difícil obtenção. No milho, a principal praga de grãos é o Sitophilus zeamais (Coleoptera: Curculionidae), devido a sua capacidade de atacar grãos tanto no campo quanto em silos. Contudo, as estimativas dos componentes aditivo e de dominância envolvidos na herança dessa resistência podem estar viesadas pela presença do efeito da epistasia. Utilizando o delineamento triple testcross, os objetivos deste trabalho foram: (i) verificar a presença da epistasia para os caracteres relacionados à resistência do milho ao S. zeamais; (ii) estimar os efeitos epistáticos em cada planta F2; e (iii) estimar o efeito da interação epistasia x ambientes para estes caracteres. As 300 progênies de retrocruzamentos utilizadas nesse estudo foram avaliadas em dois ambientes no município de Piracicaba/SP, em delineamento alfa-látice 15 x 20, no esquema fatorial com duas repetições por ambiente. Os caracteres avaliados foram: número de insetos mortos (NM); número de insetos emergidos (EM); tempo médio de desenvolvimento dos insetos (TM); índice de suscetibilidade (IS) e perda percentual de massa seca dos grãos (PE). No ambiente E. E. Anhumas a presença da epistasia foi detectada para todos os caracteres; porém, no ambiente Caterpillar o efeito da epistasia não foi detectado para nenhum dos caracteres. Na análise conjunta, os efeitos epistáticos foram detectados para os caracteres NM, EM, IS e PE. O efeito da epistasia do tipo aditivo x dominante e/ou dominante x dominante foi mais importante para todos os caracteres que a epistasia do tipo aditiva x aditiva. A interação da epistasia com os ambientes foi significativa apenas para os caracteres TM e PE. Identificaram-se efeitos epistáticos bidirecionais significativos em plantas F2 para todos os caracteres no ambiente E. E. Anhumas e para os caracteres NM, EM, IS e PE na análise conjunta. O grande número de plantas F2 que apresentaram epistasia para mais de um caráter simultaneamente sugere a presença de epistasia pleiotrópica. As estimativas da variância aditiva e da interação aditiva com ambientes foram significativamente maiores que as das variâncias de dominância para a maioria dos caracteres. As magnitudes das estimativas dos coeficientes de herdabilidade para todos os caracteres variaram de baixas a medianas. A alta correlação genética entre os caracteres EM e PE sugere que o caráter PE, que é de difícil avaliação, pode ser selecionado indiretamente através do caráter EM que é de fácil avaliação para programas de melhoramento visando resistência ao S. zeamais. Os resultados obtidos na análise conjunta sugerem que, na população estudada, a epistasia constitui um componente importante da variância genética, de forma que as estimativas da variância aditiva, de dominância, graus médios de dominância e coeficientes de herdabilidade estão viesadas. / Considered one of the most complex components in quantitative genetics, the epistasis has been ignored by plant breeders, especially in inheritance studies of plant resistance, because the traits are laborious to evaluate. Sitophilus zeamais (Coleoptera: Curculionidae) is an important grain pest, due to its ability to attack in both field and silo conditions. However, the estimation of additive and dominance components in the inheritance of resistance to this pest may be biased due to epistatic effects. With the use of the triple test cross design, this research was aimed to: (i) verify the role of epistasis in the inheritance of maize resistance to S. zeamais, (ii) estimate the epistatic effects in the resistance traits of each F2 plant; (iii) estimate the epistasis x environment interaction. The 300 backcross progenies of this study were evaluated in two environments at Piracicaba, Brazil, in 2008/2009 growing season, using an alpha-lattice 15 x 20 design in a factorial arrangement with two replications per environment. The recorded traits were: number of dead weevils (NDW); number of emerged weevils (EW); mean development period (DP); index of susceptibility (IS) and the percentage of dry grain weight loss (DGWL). The epistatic effects were detected in Anhumas Experimental Station (AES) environment for all traits although they were absent in Caterpillar Experimental Station environment. In the combined analysis epistasis was detected for NDW, EW, IS and DGWL traits. The additive x dominance and/or dominance x dominance epistasis were more important than the additive x additive epistasis for all traits. The epistasis x environment interaction was significant only for traits DP and DGWL. Significant epistatic effects, which were not unidirectional, were detected in F2 plants for all traits in AES and for the traits NDW, EW, IS and DGWL in the combined analysis. Several F2 plants presented epistasis for more than one trait simultaneously suggesting the presence of pleiotropic epistasis. Estimates of additive, dominance and the additive by environment interaction variances were significant for all traits. Estimates of additive and additive by environment interaction variances were significantly higher than those of dominance variance for most of the traits. The magnitudes of the heritability coefficients estimates ranged from low to intermediate. The genetic correlation between EW and DGWL traits suggests that DGWL, which is difficult to evaluate, can be indirectly selected by the EW trait, which is easier to evaluate, in a breeding program. The results from the combined analysis suggests that, in the population studied, the epistasis is an important component of the genetic variance; therefore, estimates of additive, dominance variance, the average levels of dominance and heritability coefficients are biased.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22287号 / 理博第4601号 / 新制||理||1660(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 酒井 章子, 教授 髙林 純示, 教授 田村 実 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

abstract: Weevils are one of the most diverse groups of animals with thousands of species suspected to remain undiscovered. The Conoderinae Schoenherr, 1833 are no exception, being especially diverse and unknown in the Neotropics where they are recognizable for their unique behaviors and color patterns among weevils. Despite these peculiarities, the group has received little attention from researchers in the past century, with almost nothing known about their evolution. This dissertation presents a series of three studies that begin to elucidate the evolutionary history of these bizarre and fascinating weevils, commencing with an overview of their biology and classificatory history (Chapter 1). Chapter 2 presents the first formal cladistic analysis on the group to redefine the New World tribes Lechriopini Lacordaire, 1865 and Zygopini, Lacordaire, 1865. An analysis of 75 taxa (65 ingroup) with 75 morphological characters yielded six equally parsimonious trees and synapomorphies that are used to reconstitute the tribes, resulting in the transfer of sixteen genera from the Zygopini to the Lechriopini and four generic transfers out of the Lechriopini to elsewhere in the Conoderinae. Chapter 3 constitutes a taxonomic revision of the genus Trichodocerus Chevrolat, 1879, the sole genus in the tribe Trichodocerini Champion, 1906, which has had an uncertain phylogenetic placement in the Curculionidae but has most recently been treated in the Conoderinae. In addition to redescriptions of the three previously described species placed in the genus, twenty-four species are newly described and an identification key is provided for all recognized species groups and species. Chapter 4 quantitatively tests the similarity in color pattern among species hypothesized to belong to several different mimicry complexes. The patterns of 160 species of conoderine weevils were evaluated for 15 categorical and continuous characters. Non-metric multidimensional scaling (NMDS) is used to visualize similarity by the proximity of individual species and clusters of species assigned to a mimicry complex in ordination space with clusters being statistically tested using permutational multivariate analysis of variance (PERMANOVA). / Dissertation/Thesis / Doctoral Dissertation Evolutionary Biology 2019

No description available.

Objetivou-se, no presente estudo, avaliar a ação de extratos orgânicos e frações de plantas com atividade inseticida, bem como, a associação de extratos orgânicos brutos com terra de diatomácea no controle de Sitophilus zeamais Mots. (Coleoptera: Curculionidade), em milho armazenado. Os extratos foram obtidos por meio de maceração em solventes orgânicos (hexano, diclorometano e etanol), em ordem crescente de polaridade, das seguintes estruturas e espécies vegetais: ramos, folhas e sementes de Annona montana e de A. mucosa; ramos de Aristolochia cf. paulistana e folhas e ramos de Casearia sylvestris. No screening visando à identificação dos extratos mais promissores, verificou-se que os extratos de sementes de A. mucosa e de A. montana, em hexano e diclorometano, seguido pelos extratos de folhas das mesmas espécies, em hexano, foram os tratamentos mais promissores, os quais foram, então, selecionados para os ensaios seguintes. Entre os extratos promissores, os obtidos de sementes de A. mucosa em hexano e em diclorometano foram os que apresentaram os menores valores de concentração letal média (CL50) (110,28 e 149,79 ppm, respectivamente). Observou-se, ainda, pequena variação no tempo letal médio (TL50) (4,14 a 6,10 dias) dos extratos selecionados. Os extratos de sementes de A. mucosa, tanto em hexano como em diclorometano, apresentaram efeito de contato sobre S. zeamais, via aplicação tópica, entretanto, nenhum dos extratos selecionados apresentou efeito inseticida via contato em superfície contaminada (papel filtro). Verificou-se, ainda, que os extratos em hexano e em diclorometano de sementes de A. mucosa apresentam pequena redução na atividade inseticida após 56 dias de armazenamento, sendo estes os extratos com maior efeito residual quando aplicados em grãos de milho visando ao controle de S. zeamais. Na associação de terra de diatomácea com extratos de sementes de A. mucosa, em hexano, e de A. montana, em diclorometano, constatou-se efeito antagônico entre as duas técnicas, quando avaliada a mortalidade dos gorgulhos expostos a tais tratamentos. Posteriormente, com base nos resultados dos ensaios biológicos e na avaliação do perfil cromatográfico dos extratos brutos obtidos por cromatografia em camada delgada (CCD), selecionaram-se os extratos em hexano de sementes de A. mucosa e de A. montana para realização de fracionamentos biomonitorados com a finalidade de se obterem os compostos responsáveis pela bioatividade observada. As frações em diclorometano e em hidroálcool do extrato em hexano de A. montana e de A. mucosa, obtidas por partição líquido-líquido, apresentam efeito inseticida para S. zeamais, sem, contudo, haver diferença entre ambas as frações de cada extrato. Todas as frações bioativas, de ambos os extratos, foram analisadas por CCD, utilizando-se diferentes eluentes e reveladores, por cromatografia líquida de alta eficiência (CLAE) e por ressonância magnética nuclear de hidrogênio (RMN de 1H), e seus resultados mostraram a presença de alcalóides e acetogeninas em todas as amostras analisadas. Assim, possivelmente, compostos de tais classes químicas estão relacionados com a bioatividade observada. / The aim of the present study was to evaluate the effects of organic extracts and fractions of plants with insecticidal activity, as well as, the combination of crude organic extracts with diatomaceous earth in order to control Sitophilus zeamais Mots. (Coleoptera: Curculionidade) in stored corn. The extracts were obtained by maceration technique using organic solvents (hexane, dichloromethane and ethanol) respecting the order of increasing polarity. The species and their structures used were: branches, leaves and seeds of Annona montana and A. mucosa; branches of Aristolochia cf. paulistana and branches and leaves of Casearia sylvestris. In the screening assay, the most promising treatments were extracts from seeds of A. mucosa and A. montana, in hexane and dichloromethane, followed by extracts from leaves of the same species, in hexane. Thus, these extracts were selected for further tests. Among the promising extracts, the seed extract of A. mucosa in hexane and in dichloromethane showed the lowest Lethal Concentration (LC50) (110.28 and 149.79 ppm, respectively). There was a little variation in the Lethal Time (LT50) of the selected extracts ranged between 4.14 and 6.10 days. Seed extracts of A. mucosa, in hexane and dichloromethane, showed a contact effect to S. zeamais via topical application; althought, none of the selected extracts showed insecticidal effects by contact in contaminated surface (filter paper). Both hexane and dichloromethane extracts from seeds of A. mucosa showed a slight decrease in their insecticidal activity after 56 days being these values the highest residual effect among the extracts tested when applied on maize for the control of S. zeamais. The association of diatomaceous earth with A. mucosa seed extracts in hexane, as well as, A. montana in dichloromethane caused an antagonistic effect between the two techniques when evaluated the mortality of weevils exposed to such treatments. Supported by the results of biological tests and the evaluation of the chromatographic profile of crude extracts obtained by thin layer chromatography (TLC), we selected the hexanic extracts of A. mucosa and A. montana seeds to fractionation and identify the responsible compounds by the observed bioactivity. The hydroalcoholic and dichloromethanic fractions of the hexane extract of A. montana and A. mucosa, obtained by liquid-liquid partition, showed insecticidal effects on S. zeamais having no difference among them about the provoked mortalities. All the bioactive fractions of both extracts were analyzed with TLC, using different solvents and developers, by High Performance Liquid Chromatography (HPLC) and Proton Nuclear Magnetic Resonance (1H NMR), and their results showed alkaloids and acetogenins in all samples. Thus, possibly, compounds of these chemical classes may be related to the bioactivity observed.

Objetivou-se, no presente estudo, avaliar a ação de extratos orgânicos e frações de plantas com atividade inseticida, bem como, a associação de extratos orgânicos brutos com terra de diatomácea no controle de Sitophilus zeamais Mots. (Coleoptera: Curculionidade), em milho armazenado. Os extratos foram obtidos por meio de maceração em solventes orgânicos (hexano, diclorometano e etanol), em ordem crescente de polaridade, das seguintes estruturas e espécies vegetais: ramos, folhas e sementes de Annona montana e de A. mucosa; ramos de Aristolochia cf. paulistana e folhas e ramos de Casearia sylvestris. No screening visando à identificação dos extratos mais promissores, verificou-se que os extratos de sementes de A. mucosa e de A. montana, em hexano e diclorometano, seguido pelos extratos de folhas das mesmas espécies, em hexano, foram os tratamentos mais promissores, os quais foram, então, selecionados para os ensaios seguintes. Entre os extratos promissores, os obtidos de sementes de A. mucosa em hexano e em diclorometano foram os que apresentaram os menores valores de concentração letal média (CL50) (110,28 e 149,79 ppm, respectivamente). Observou-se, ainda, pequena variação no tempo letal médio (TL50) (4,14 a 6,10 dias) dos extratos selecionados. Os extratos de sementes de A. mucosa, tanto em hexano como em diclorometano, apresentaram efeito de contato sobre S. zeamais, via aplicação tópica, entretanto, nenhum dos extratos selecionados apresentou efeito inseticida via contato em superfície contaminada (papel filtro). Verificou-se, ainda, que os extratos em hexano e em diclorometano de sementes de A. mucosa apresentam pequena redução na atividade inseticida após 56 dias de armazenamento, sendo estes os extratos com maior efeito residual quando aplicados em grãos de milho visando ao controle de S. zeamais. Na associação de terra de diatomácea com extratos de sementes de A. mucosa, em hexano, e de A. montana, em diclorometano, constatou-se efeito antagônico entre as duas técnicas, quando avaliada a mortalidade dos gorgulhos expostos a tais tratamentos. Posteriormente, com base nos resultados dos ensaios biológicos e na avaliação do perfil cromatográfico dos extratos brutos obtidos por cromatografia em camada delgada (CCD), selecionaram-se os extratos em hexano de sementes de A. mucosa e de A. montana para realização de fracionamentos biomonitorados com a finalidade de se obterem os compostos responsáveis pela bioatividade observada. As frações em diclorometano e em hidroálcool do extrato em hexano de A. montana e de A. mucosa, obtidas por partição líquido-líquido, apresentam efeito inseticida para S. zeamais, sem, contudo, haver diferença entre ambas as frações de cada extrato. Todas as frações bioativas, de ambos os extratos, foram analisadas por CCD, utilizando-se diferentes eluentes e reveladores, por cromatografia líquida de alta eficiência (CLAE) e por ressonância magnética nuclear de hidrogênio (RMN de 1H), e seus resultados mostraram a presença de alcalóides e acetogeninas em todas as amostras analisadas. Assim, possivelmente, compostos de tais classes químicas estão relacionados com a bioatividade observada. / The aim of the present study was to evaluate the effects of organic extracts and fractions of plants with insecticidal activity, as well as, the combination of crude organic extracts with diatomaceous earth in order to control Sitophilus zeamais Mots. (Coleoptera: Curculionidade) in stored corn. The extracts were obtained by maceration technique using organic solvents (hexane, dichloromethane and ethanol) respecting the order of increasing polarity. The species and their structures used were: branches, leaves and seeds of Annona montana and A. mucosa; branches of Aristolochia cf. paulistana and branches and leaves of Casearia sylvestris. In the screening assay, the most promising treatments were extracts from seeds of A. mucosa and A. montana, in hexane and dichloromethane, followed by extracts from leaves of the same species, in hexane. Thus, these extracts were selected for further tests. Among the promising extracts, the seed extract of A. mucosa in hexane and in dichloromethane showed the lowest Lethal Concentration (LC50) (110.28 and 149.79 ppm, respectively). There was a little variation in the Lethal Time (LT50) of the selected extracts ranged between 4.14 and 6.10 days. Seed extracts of A. mucosa, in hexane and dichloromethane, showed a contact effect to S. zeamais via topical application; althought, none of the selected extracts showed insecticidal effects by contact in contaminated surface (filter paper). Both hexane and dichloromethane extracts from seeds of A. mucosa showed a slight decrease in their insecticidal activity after 56 days being these values the highest residual effect among the extracts tested when applied on maize for the control of S. zeamais. The association of diatomaceous earth with A. mucosa seed extracts in hexane, as well as, A. montana in dichloromethane caused an antagonistic effect between the two techniques when evaluated the mortality of weevils exposed to such treatments. Supported by the results of biological tests and the evaluation of the chromatographic profile of crude extracts obtained by thin layer chromatography (TLC), we selected the hexanic extracts of A. mucosa and A. montana seeds to fractionation and identify the responsible compounds by the observed bioactivity. The hydroalcoholic and dichloromethanic fractions of the hexane extract of A. montana and A. mucosa, obtained by liquid-liquid partition, showed insecticidal effects on S. zeamais having no difference among them about the provoked mortalities. All the bioactive fractions of both extracts were analyzed with TLC, using different solvents and developers, by High Performance Liquid Chromatography (HPLC) and Proton Nuclear Magnetic Resonance (1H NMR), and their results showed alkaloids and acetogenins in all samples. Thus, possibly, compounds of these chemical classes may be related to the bioactivity observed.

All previous taxonomic studies on the Ectemnorhinus group of weevils have been based primarily on morphological data. While these studies are invaluable, some questions can only be addressed adequately through molecular studies. This is especially true when studying the genetic relationships and phylogeograpic patterns of taxa endemic to the South Indian Ocean Province (SIP) biotas that have long been controversial. The Ectemnorhinus group of genera is a monophyletic unit of weevils endemic to the region. The present study focused mainly on the Ectemnorhinus group of weevils found on the Prince Edward Islands archipelago (PEIA). The mitochondrial cytochrome oxidase I gene was targeted when investigating relationships among members of this weevil group. On the PEIA, it is important to note that Marion Island (MI) and Prince Edward Island (PEI) differ in terms of alien invasive species, such as the introduced house mouse Mus musculus and in conservation management strategies. Since emergence, a series of volcanic and glaciation events have occurred on Marion Island, whilst Prince Edward Island has remained largely unaffected by glaciation. Phylogenetic analyses revealed the presence of two genetically and morphometrically distinct species of Ectemnorhinus weevils on PEI, whilst evidence for a single species, comprising diverse genetically discrete populations was found on MI. Based on these results, the species unique to PEI has been designated E. kuscheli n. sp., whilst the present study confirmed the synonymy between E. similis and E. marioni, the two species originally described from MI. Ectemnorhinus kucheli appears to be restricted to PEI, whereas E. similis occurs on both MI and PEI. When investigating the population dynamics of the Ectemnorhinus weevils on the PEIA, the data indicated that PEI was the first of the two islands of the PEIA to be colonized by Ectemnorhinus weevils, at an estimated time of coalescence of approximately 0.3116 million years ago (MYA). The PEI population then acted as the source population for the colonization of MI by Ectemnorhinus weevils some time before the last glaciation, approximately 10 000 to 35 000 years ago. The separation by distance of the PEI Ectemnorhinus weevils from those on MI then gave rise to two species by allopatric speciation on MI. During the last glaciations, MI was extensively glaciated with only the southwestern corner of the island being free of ice. This extensive glaciation of MI would have resulted in the eradication of all E. similis on MI except for those occurring on the ice-free southwestern corner of the island. At the end of the last glacial maximum, when the ice started to melt, the coastal areas of MI emerged first from beneath the ice and were available for re-colonization by weevils. The movement of weevils that were isolated in the south-western corner of MI, along the coastal areas of the island, was assisted by strong, frequent south-western winds. Subsequent, post-glacial volcanism during the Holocene was then responsible for the fragmentation of the new migrants, resulting in small population pockets surrounded by fresh, uninhabitable lava and subsequent divergence of each populations. When the Holocene black lava became re-colonizeable, the weevils from the different isolated populations migrated to the remainder of the island. Currently, members of the different genetically-identified populations occur in sympatry and in some cases even on the same plant, but no noticeable geneflow was detected between them. It is thus suggested that the time of isolation, before the post-glacial black lava during Holocene became hospitable, was sufficiently long and the populations sufficiently small that a number of genetically-discrete populations arose. Consequently, the present study recognises two genetically discrete populations of E. kucheli on PEI and seven discrete E. similis populations on MI that are morphologically indistinct. When examining the relationships among 13 species from five different islands within the South Indian Ocean Province (SIP) that are representative of 22 populations within the genera Palirhoeus, Bothrometopus and Ectemnorhinus, there was little support for separating the genus Palirhoeus from Bothrometopus, and no support for the morphologically-delineated species groups currently recognized within Bothrometopus. The present study shows that colonization of the Prince Edward Islands is likely to have occurred repeatedly from other islands within the SIP and that Bothrometopus parvulus on the PEIA comprises two species that are not sister taxa. The second novel con-generic species was therefore designated Bothrometopus huntleyi n. sp. and examination of the genetically identified specimens resulted in the indentification of distinguishing morphological characteristics. The analyses indicated that B. huntleyi arose approximately 0.5 million years ago from a high-altitude population that is still present on MI. The first major intra- and inter-island dispersal event occurred ~0.338 MYA, coinciding with the glaciation-free second volcanic stage on MI. Apart from this early inter-island colonisation, only one other between-island dispersal event, corresponding with the glaciation-free seventh volcanic stage, was detected. Genetically discrete weevil complexes on each of the islands of the PEIA together with the low levels of inter-island gene flow reaffirm the need to control alien invasive mice, which are restricted to MI, and which prey on these weevil species. / Thesis (PhD)--University of Pretoria, 2012. / Zoology and Entomology / unrestricted